Summary: In addition to its recognized actions on water conservation and blood pressure, the neuropeptide vasopressin is released into the pituitary portal circulation and within distinct brain areas during stressful challenges. The neuroendocrine effects of vasopressin are mediated by specific receptor of subtype V1b in the anterior pituitary and V1a and V1b subtypes in the brain. Vasopressin produced by parvocellular neurons of the PVN potentiates the stimulatory effect of CRH on pituitary ACTH secretion acting through plasma membrane receptors of the V1b subtype (V1bR). During prolonged activation of the HPA axis there is a predominant increase in VP in parvocellular hypothalamic neurons and pituitary V1b receptors compared with CRH and CRH receptors, suggesting that VP becomes the main mediator of pituitary corticotroph responsiveness during chronic stress. To test this hypothesis, we examined the effect of pharmacologic VP receptor blockade on the ACTH and corticosterone responses of 14-day repeatedly restrained rats. In spite of the increased vasopressinergic activity, repeatedly restrained rats showed lower ACTH and corticosterone responses to 10 min white noise compared with handled controls. These responses were unchanged by the non-peptide selective V1b receptor antagonist, SSR149415, 10 &#61549;g, i.v., 1h before noise application. In contrast to the response to noise stress, plasma ACTH responses to i.p. hypertonic saline injection were enhanced in the repeatedly restrained rats compared with handled controls but responses were also unaffected by SSR149415 (30mg/kg/orally) administered daily 1 h before restraint. Since SSR149415 bioactivity, assessed by corticosterone responses to exogenous VP, was low, we used minipump infusion of the peptide V1 receptor antagonist, dGlyPhaa1,D-Tyr(et), Lys, ArgVP (V1-Ant) for 14 days, which effectively blocked ACTH responses to exogenous VP. Chronic V1-Ant infusion significantly reduced plasma ACTH responses to i.p. hypertonic saline in handled controls but not in repeatedly restrained rats. These data show that VP contributes to the ACTH responses to an acute stressor but that increased vasopressinergic activity is not responsible for the enhanced ACTH responses to a novel stress.[unreadable] [unreadable] The little impact of vasopressinergic blockade on HPA axis activity during chronic stress, suggests that VP has additional roles, such as controlling the number of pituitary corticotrophs. The role of VP mediating pituitary corticotroph mitogenesis was studied in adrenalectomized rats by examining the effect of a peptide VP V1 receptor antagonist (V1-ant) on the number of cells incorporating bromouridine (BrdU). Long term adrenalectomy increased the number of both BrdU labeled cells and ACTH stained cells. Osmotic minipump infusion of V1-ant for 28 days prevented adrenalectomy-induced increases in BrdU incorporation but not the changes in the number of ACTH-stained cells. Unexpectedly, co-localization of BrdU uptake in ACTH positive cells was minor and this was unaffected by adrenalectomy or V1-antagonist infusion. No BrdU stained nuclei were observed in LH, TSH, PRL, GH, folliculo-stellate cells or nestin-labeled progenitor cells. The pituitary corticotroph exclusive transcription factor, Tpit, colocalized in >80% of ACTH containing cells but only in 5% of BrdU labeled nuclei in controls and 10% in adrenalectomized rats. In V1bR knock out mice, the number of cells incorporating BrdU following adrenalectomy was lower than in wild type, and there was no major co-localization of BrdU and ACTH. The data demonstrates that VP mediates mitogenic activity in the pituitary during long term adrenalectomy. The lack of colocalization of ACTH and BrdU, suggest that recruitment of corticotrophs during adrenalectomy occurs from undifferentiated cells. This data suggest that one of the functions of the marked increases in parvocellular vasopressinergic activity during adrenalectomy (and probably chronic stress) is regulating cell proliferation and remodeling of the pituitary tissue.[unreadable] [unreadable] Based on the observation that VP prevented serum deprivation-induced cell death in the neuronal cell line, H32, which expresses endogenous V1 receptors, we tested the hypothesis that VP has anti-apoptotic properties. Flow cytometry experiments showed that 10nM VP prevented serum deprivation-induced cell death and annexin V binding. Serum deprivation increased caspase-3 activity in a time and serum concentration dependent manner, and VP prevented these effects in a V1 receptor-specific manner. The signaling pathways mediating the anti-apoptotic effect of VP involve mitogen activated protein (MAP) kinase MAPK and extracellular signal-regulated kinases (ERK), Ca2+/calmodulin dependent kinase (CaMK) and protein kinase C (PKC). Western blot analyses revealed time-dependent decreases of BAD phosphorylation and increases in cytosolic levels of cytochrome c following serum deprivation, effects prevented by 10nM VP. These data demonstrate that activation of endogenous V1 VP receptors prevents serum deprivation-induced apoptosis, through phosphorylation-inactivation of the pro-apoptotic protein, BAD, and consequent decreases in cytosolic cytochome c and caspase-3 activation. The data suggest that VP has anti-apoptotic activity in neurons and that VP may act as a neuroprotective agent in the brain.